R. van der Zee

The Human Endoplasmic Reticulum Molecular Chaperone BiP Is an Autoantigen for Rheumatoid Arthritis and Prevents the Induction of Experimental Arthritis

Rheumatoid arthritis (RA) is the most common, crippling human autoimmune disease. Using Western blotting and tandem mass spectroscopy, we have identified the endoplasmic reticulum chaperone BiP, a 78-kDa glucose-regulated protein, as a possible autoantigen. It preferentially stimulated increased proliferation of synovial T cells from patients with RA but not from patients with other arthritides. Mice with established collagen- or pristane-induced arthritis developed IgG Abs to BiP. Although BiP injected in CFA failed to induce arthritis in several strains of rats and mice, including HLA-DR4+/−- and HLA-DR1+/+-transgenic animals, it completely inhibited the development of arthritis when given i.v. 1 wk before the injection of type II collagen arthritis. Preimmunization with BiP suppressed the development of adjuvant arthritis in Lewis rats in a similar manner. This is the first report of a mammalian chaperone that is an autoantigen in human RA and in experimental arthritis and that can also prevent the induction of experimental arthritis. These findings may stimulate the development of new immunotherapies for the treatment of RA.

Induction of Oral Tolerance to Oxidized Low-Density Lipoprotein Ameliorates Atherosclerosis

Background— Oxidation of low-density lipoprotein (LDL) and the subsequent processing of oxidized LDL (oxLDL) by macrophages results in activation of specific T cells, which contributes to the development of atherosclerosis. Oral tolerance induction and the subsequent activation of regulatory T cells may be an adequate therapy for the treatment of atherosclerosis. Methods and Results— Tolerance to oxLDL and malondialdehyde-treated LDL (MDA-LDL) was induced in LDL receptor−/− mice fed a Western-type diet by oral administration of oxLDL or MDA-LDL before the induction of atherogenesis. Oral tolerance to oxLDL resulted in a significant attenuation of the initiation (30% to 71%; P<0.05) and progression (45%; P<0.05) of atherogenesis. Tolerance to oxLDL induced a significant increase in CD4+CD25+Foxp3+ cells in spleen and mesenteric lymph nodes, and these cells specifically responded to oxLDL with increased transforming growth factor-&bgr; production. Tolerance to oxLDL also increased the mRNA expression of Foxp3, CTLA-4, and CD25 in the plaque. In contrast, tolerance to MDA-LDL did not affect atherogenesis. Conclusions— OxLDL-specific T cells, present in LDL receptor−/− mice and important contributors in the immune response leading to atherosclerotic plaque, can be counteracted by oxLDL-specific CD4+CD25+Foxp3+ regulatory T cells activated via oral tolerance induction to oxLDL. We conclude that the induction of oral tolerance to oxLDL may be a promising strategy to modulate the immune response during atherogenesis and a new way to treat atherosclerosis.

Induction of Oral Tolerance to HSP60 or an HSP60-Peptide Activates T Cell Regulation and Reduces Atherosclerosis

Objective—HSP60-specific T cells contribute to the development of the immune responses in atherosclerosis. This can be dampened by regulatory T cells activated via oral tolerance induction, and we explored the effect of oral tolerance induction to HSP60 and the peptide HSP60 (253 to 268) on atherosclerosis. Methods and Results—HSP60 and HSP60 (253 to 268) were administered orally to LDLr−/− mice before induction of atherosclerosis and resulted in a significant 80% reduction in plaque size in the carotid arteries and in a 27% reduction in plaque size at the aortic root. Reduction in plaque size correlated with an increase in CD4+CD25+Foxp3+ regulatory T cells in several organs and in an increased expression of Foxp3, CD25, and CTLA-4 in atherosclerotic lesions of HSP60-treated mice. The production of interleukin (IL)-10 and transforming growth factor (TGF)-&bgr; by lymph node cells in response to HSP60 was observed after tolerance induction. Conclusion—Oral tolerance induction to HSP60 and a small HSP60-peptide leads to an increase in the number of CD4+CD25+Foxp3+ regulatory T cells, resulting in a decrease in plaque size as a consequence of increased production of IL-10 and TGF-&bgr;. We conclude that these beneficial results of oral tolerance induction to HSP60 and HSP60 (253 to 268) may provide new therapeutic approaches for the treatment of atherosclerosis.

Experimental immunization with anti-rheumatic bacterial extract OM-89 induces T cell responses to heat shock protein (hsp)60 and hsp70; modulation of peripheral immunological tolerance as its possible mode of action in the treatment of rheumatoid arthritis (RA)

OM‐89 is a bacterial (Escherichia coli) extract used for oral administration in the treatment of RA. Given the evidence that immunity to bacterial heat shock antigens plays a critical role in the immunomodulation of arthritis and possibly inflammation in general, the purpose of the present studies was to evaluate the presence and immunogenicity of hsp in OM‐89. Furthermore, we studied the effects of OM‐89 in an experimental arthritis, where hsp are known to have a critical significance in disease development. In rats immunization with OM‐89 was found to lead to proliferative T cell responses to hsp60 and hsp70 of both E. coli and mycobacterial origin. Conversely, immunization with hsp antigens was also found to induce T cell reactivity specific for OM‐89. Based on this and the antigen specificity analysis of specific T cell lines, hsp70 (DnaK) turned out to be one of the major immunogenic constituents of OM‐89. Parenteral immunization with OM‐89 was found to reduce resistance to adjuvant arthritis (AA), whereas oral administration was found to protect against AA. Given the arthritis‐inhibitory effect of oral OM‐89 in AA, it is possible that peripheral tolerance is induced at the level of regulatory T cells with specificity for hsp. This may also constitute a mode of action for OM‐89 as an arthritis‐suppressive oral drug.

In vitro T lymphocyte responses to proteinase 3 (PR3) and linear peptides of PR3 in patients with Wegener's granulomatosis (WG)

T cell‐mediated immunity is thought to play an important role in the pathogenesis of WG. In previous studies a minority of WG patients as well as some healthy controls showed in vitro proliferation of their peripheral blood mononuclear cells (PBMC) to PR3, the main autoantigen in WG. The relevant peptides responsible for this in vitro proliferation have not been identified. In order to define immunogenic peptides, PBMC of 13 WG patients in remission and 10 healthy controls were tested for proliferation to linear peptides of PR3 and to whole PR3. Fifty overlapping peptides spanning the whole PR3 sequence were synthesized. Peptides were tested in pools of five peptides and as single peptide. PBMC of two WG patients and one healthy control proliferated to whole PR3 and to peptide pools. In addition, 10 WG patients and eight healthy controls that did not proliferate to whole PR3 did proliferate to pools of PR3 peptides. Although more WG patients tended to react to particular peptide pools, no significant difference was seen between lymphocyte proliferation to PR3 peptides of WG patients and that of healthy controls. The pools of peptides recognized were mainly located at the N‐ and C‐terminus of PR3. No correlation was observed between HLA type and proliferation on particular peptide pools. No proliferation of PBMC was observed to single peptides. In conclusion, T cells of WG patients proliferate in vitro more frequently to PR3 peptides than to the whole PR3 protein. Peptides derived from the signal sequence, the propeptide or peptides located at the C‐terminus of PR3 induce highest levels of proliferation. No specific PR3 sequence could be identified that was preferentially recognized by PBMC of WG patients compared with controls.

Stimulation of synovial fluid mononuclear cells with the human 65-kD heat shock protein or with live enterobacteria leads to preferential expansion of TCR-γδ+ lymphocytes

T lymphocyte responses to heterologous or self 65‐kD heat shock protein (hsp) have been implicated in the pathogenesis of various forms of arthritis. To delineate the relationship of 65‐kD hsp to different synovial fluid (SF) T cell subsets, we stimulated synovial fluid (SFMC) and peripheral blood mononuclear cells (PBMC) from patients with different inflammatory rheumatic diseases and from healthy controls with human or mycobacterial 65‐kD hsp, tetanus toxoid (TT), heat‐killed or live Yersinia enterocotitica. Phenotyping of the resulting T cell lines revealed an increase of up to 97% TCR‐γδ+ lymphocytes in the 65‐kD hsp‐stimulatcd SF‐derived lines. This expansion of TCR‐γδ+ cells was less pronounced with cultures of PBMC. A preferential expansion of TCR‐γδ+ cells was also shown after SFMC stimulation with live, but not with heat‐killed Yersinia or with TT. We conclude that a common mechanism is involved in the selective expansion of TCR‐γδ+ lymphocytes upon SFMC infection with live Yersinia or upon contact with 65‐kD hsp. Out of a panel of TCR‐γδ+ T lymphocyte clones (TLC) derived from a human 65‐kD hsp‐stimulated line, only a minority of TLC proliferated weakly upon restimulation with this antigen in the presence of autologous monocytes, whereas TCR‐αβ+ TLC responded vigorously to the human 65‐kD hsp and in some cases also cross‐recognized the mycobacterial hsp homologue and/or heat‐killed Yersinia. This implies that additional factors or cells may be present in the milieu of SFMC cultures that propagate the expansion of TCR‐γδ+ cells in response to 65‐kD hsp or live bacteria.

Balancing the immune system: Th1 and Th2

CD4+ T cells are subdivided into Th1 and Th2 cells. Their relative presence or activation is thought to have a regulatory effect on immune behaviour. Until recently, the relative suppression of Th1 cells by the relative increase of Th2 activities, was thought to be a main mechanism of keeping or restoring the balance in a diseased immune system. Nowadays, however, a specialised subset of regulatory T cells is held to be responsible for the main effects of securing a balanced immune system. It is possible that heat shock proteins (hsps) are relevant antigens driving such regulation. Heat shock proteins are known to be immunodominant antigens of bacteria. They are evolutionarily strongly conserved proteins present in all eukaryotic and prokaryotic cellular organisms and are upregulated by several forms of stress. Despite (the paradigm of) self tolerance, hsp-epitopes homologous to endogenous host hsp sequences have been implicated as T cell epitopes to endow cross reactive, hsp specific T cells with the capacity to regulate inflammation, such as in experimentally induced autoimmune diseases. Such T cells were found to produce regulatory cytokines like IL10, in contrast with T cells induced with other conserved microbial proteins that are not upregulated by stress. Hsps have been implicated in immune regulation not only as upregulated targets of adaptive immunity during inflammatory stress, but recently also as triggering factors for innate immunity through activation via Toll-like receptors (TLRs). Th1 cells or proinflammatory T cells are known to produce cytokines with proinflammatory activities. Therefore they are supposed to be critically involved in inflammatory conditions such as autoimmune arthritis. Th2 or helper T cells are known to produce cytokines that help B cells to become activated and to switch their class of antibody. Some of the cytokines produced by Th2 cells (IL4, IL5, IL10, and IL13) also have immune …

Heat shock proteins are no DAMPs, rather 'DAMPERs'

Originally, the immune system was seen as a system that primarily combats infection. But, as discussed in the recent Review article by Grace Chen and Gabriel Nuñez (Sterile inflammation: sensing and reacting to damage. Nature Rev. Immunol. 10, 826–837 (2010))1, most of us accept the idea that the immune system is key to tissue homeostasis and even to homeostatic interactions with the outside antigenic world, including the gut microbiota1,2. However, much remains unknown about the nature of the triggers of pro-inflammatory innate immune responses. In a broad sense, two main types of activators prevail: non-self antigens (known as pathogen-associated molecular patterns (PAMPs)), which are present in or released from infectious invaders; and damageassociated molecular patterns (DAMPs), which are host molecules (such as highmobility group box 1 protein (HMGB1)) released from damaged cells under necrotic but not apoptotic conditions. Heat shock proteins (HSPs) are frequently mentioned as prime examples of DAMPs (see, for example, REFS 1,3). There are, however, several qualities inherent to HSPs that disqualify them as DAMPs. First, DAMPs are intracellular molecules normally hidden from recognition by the immune system, whereas HSPs are freely present in the extracellular fluids4,5 and are also frequently exposed at the outer surface of cells (both eukaryotic and prokaryotic cells)6,7. Second, Toll-like receptor 2 (TLR2) and TLR4 are seen as two of the main receptors involved in the recognition of HSP60 and HSP70. These HSPs are released from cells under necrotic conditions, but it has been shown that TLR2 and TLR4 are not required for the host response to DAMPs that are derived from necrotic cells8. Moreover, other receptors for HSPs, such as the HSP70 scavenger receptor SRA1, have been shown to confer a suppressive rather than an activating signal to host cells9. Third, in vitro-cultured dendritic cells have been shown to adopt a tolerizing phenotype, rather than a mature or activated phenotype, in the presence of HSPs10,11. Fourth, in experimental models of autoimmunity and of tissue or tumour transplants, immunization with HSPs was shown to lead to the induction of regulatory T cells, which suppressed disease or transplant rejection12–19. Taken together, these phenomena argue against the involvement of HSPs in the induction of the immune response to damagederived signals. On the contrary, HSPs seem to have a dampening effect on immune activation and have the capacity to promote immune homeostasis12,20. A possible reason for the proposition that HSPs are DAMPs could be that some of the early studies used recombinant HSP preparations that were contaminated with lipopolysaccharide (LPS), although in most cases contamination levels were not determined10,21. More recent studies (reviewed in REF. 5) using HSP preparations from which the contaminants had been effectively removed did not provide evidence to support a proinflammatory function for HSPs. Based on the current experimental data, it seems that HSPs are key elements in the type of immune system responsiveness or reactivity that is induced by the following three typical conditions: one, sterile tissue damage, when tissue-derived HSPs in combination with DAMPs activate tissue repair and regulation; two, damage caused by infectious pathogens, when HSPs in the presence of DAMPs and pathogen-derived PAMPs lead to a full proinflammatory response with elimination of infection and regulation; and three, homeostatic interactions with commensal symbionts of the gut microbiota, when a combination of PAMPs and HSPs leads to the regulation and maintenance of symbiosis22–24. Given the ubiquitous presence and stressinducible nature of HSPs in both tissue cells and microbial invaders or symbionts, therapeutic targeting of HSPs offers an attractive possibility for the fine-tuning of such immune responses and the dampening of inflammation through the induction or activation of regulatory T cells.

Heat shock proteins can be targets of regulatory T cells for therapeutic intervention in rheumatoid arthritis

Abstract Rheumatoid arthritis (RA) is a chronic autoimmune disease characterised by excessive immune responses resulting in inflammation of the joints. Although current therapies can be successful in dampening inflammation, a long-lived state of tolerance is seldom achieved. Therefore, novel therapies are needed that restore and maintain tolerance in patients with RA. Targeting regulatory T cells (Tregs) is a successful strategy to achieve tolerance, as was shown in studies performed in animal models and in human clinical trials. The antigen-specificity of Tregs is crucial for their effectiveness and allows for very specific targeting of these cells. However, which antigen is suitable for autoimmune diseases such as RA, for which the autoantigens are largely unknown? Heat shock proteins (HSPs) are ubiquitously expressed and can be up-regulated during inflammation. Additionally, HSPs, or HSP-derived peptides are immunogenic and can be recognised by a variety of immune cells, including Tregs. Therefore, this review highlights the potential of HSP-specific Tregs to control inflammatory immune responses. Targeting HSP-specific Tregs in RA can be achieved via the administration of HSPs (derived peptides), thereby controlling inflammatory responses. This makes HSPs attractive candidates for therapeutic intervention in chronic autoimmune diseases, with the ultimate goal of inducing long-lasting tolerance.

Mycobacterial and mouse HSP70 have immuno-modulatory effects on dendritic cells

Previously, it has been shown that heat shock protein 70 (HSP70) can prevent inflammatory damage in experimental autoimmune disease models. Various possible underlying working mechanisms have been proposed. One possibility is that HSP70 induces a tolerogenic phenotype in dendritic cells (DCs) as a result of the direct interaction of the antigen with the DC. Tolerogenic DCs can induce antigen-specific regulatory T cells and dampen pathogenic T cell responses. We show that treatment of murine DCs with either mycobacterial (Mt) or mouse HSP70 and pulsed with the disease-inducing antigen induced suppression of proteoglycan-induced arthritis (PGIA), although mouse HSP70-treated DCs could ameliorate PGIA to a greater extent. In addition, while murine DCs treated with Mt- or mouse HSP70 had no significantly altered phenotype as compared to untreated DCs, HSP70-treated DCs pulsed with pOVA (ovalbumin peptide 323–339) induced a significantly increased production of IL-10 in pOVA-specific T cells. IL-10-producing T cells were earlier shown to be involved in Mt HSP70-induced suppression of PGIA. In conclusion, this study indicates that Mt- and mouse HSP70-treated BMDC can suppress PGIA via an IL-10-producing T cell-dependent manner.